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Marvin Wickens

Publications and source records attributed to Marvin Wickens.

At least 19 recordsLinked to original sources
PubMed2006

Autoregulation of GLD-2 cytoplasmic poly(A) polymerase.

Cytoplasmic polyadenylation regulates mRNA stability and translation and is required for early development and synaptic plasticity. The GLD-2 poly(A) polymerase catalyzes cytoplasmic polyadenylation in the germline of metazoa. Among vertebrates, the enzyme is encoded by two isoforms of mRNA that differ only in the length of their 3'-UTRs. Here we focus on regulation of vertebrate GLD-2 mRNA. We show that the 3'-UTR of GLD-2 mRNA elicits its own polyadenylation and translational activation during frog oocyte maturation. We identify the sequence elements responsible for repression and activation, and demonstrate that CPEB and PUF proteins likely mediate repression in the resting oocyte. Regulated polyadenylation of GLD-2 mRNA is conserved, as are the key regulatory elements. Poly(A) tails of GLD-2 mRNA increase in length in the brain in response to neuronal stimulation, suggesting that a comparable system exists in that tissue. We propose a positive feedback circuit in which translation of GLD-2 mRNA is stimulated by its polyadenylation, thereby reinforcing the switch to polyadenylate and activate batteries of mRNAs.

3' Untranslated Regions↗
PubMed2006

PUF protein-mediated deadenylation is catalyzed by Ccr4p.

PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Binding Sites↗
PubMed2006

The GLD-2 poly(A) polymerase activates gld-1 mRNA in the Caenorhabditis elegans germ line.

mRNA regulation is crucial for many aspects of metazoan development and physiology, including regulation of stem cells and synaptic plasticity. In the nematode germ line, RNA regulators control stem cell maintenance, the sperm/oocyte decision, and progression through meiosis. Of particular importance to this work are three GLD (germ-line development) regulatory proteins, each of which promotes entry into the meiotic cell cycle: GLD-1 is a STAR/Quaking translational repressor, GLD-2 is a cytoplasmic poly(A) polymerase, and GLD-3 is a homolog of Bicaudal-C. Here we report that the gld-1 mRNA is a direct target of the GLD-2 poly(A) polymerase: polyadenylation of gld-1 mRNA depends on GLD-2, the abundance of GLD-1 protein is dependent on GLD-2, and the gld-1 mRNA coimmunoprecipitates with both GLD-2 and GLD-3 proteins. We suggest that the GLD-2 poly(A) polymerase enhances entry into the meiotic cell cycle at least in part by activating GLD-1 expression. The importance of this conclusion is twofold. First, the activation of gld-1 mRNA by GLD-2 identifies a positive regulatory step that reinforces the decision to enter the meiotic cell cycle. Second, gld-1 mRNA is initially repressed by FBF (for fem-3 binding factor) to maintain stem cells but then becomes activated by the GLD-2 poly(A) polymerase once stem cells begin to make the transition into the meiotic cell cycle. Therefore, a molecular switch regulates gld-1 mRNA activity to accomplish the transition from mitosis to meiosis.

Adenosine↗
PubMed2006

A three-hybrid screen identifies mRNAs controlled by a regulatory protein.

RNA-protein interactions are important in many biological contexts. Identification of the networks that connect regulatory proteins to one another and to the mRNAs they control is a critical need. Here, we use a yeast three-hybrid screening approach to identify RNAs that bind a known RNA regulatory protein, the Saccharomyces cerevisiae PUF protein, Mpt5p. The assay selects RNAs that bind in vivo using simple phenotypes and reporter genes. It enables rapid analyses of the affinity and specificity of the interaction. We show that the method identifies mRNAs that are genuinely regulated by the protein in vivo, and that it complements biochemical strategies, yielding a set of mRNAs that overlap with, but are distinct from, those obtained by biochemical means. The approach we describe facilitates construction of protein-RNA linkage maps.

Biological Assay↗
PubMed2006

PUF proteins bind Pop2p to regulate messenger RNAs.

PUF proteins, a family of RNA-binding proteins, interact with the 3' untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. Here, we focus on how PUF proteins enhance deadenylation and mRNA decay. We show that a yeast PUF protein physically binds Pop2p, which is a component of the Ccr4p-Pop2p-Not deadenylase complex, and that Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. We reconstitute regulated deadenylation in vitro and demonstrate that the PUF-Pop2p interaction is conserved in yeast, worms and humans. We suggest that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins.

3' Untranslated Regions↗
PubMed2005

LIP-1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans.

Caenorhabditis elegans germline cells are maintained in an undifferentiated and mitotically dividing state by Notch signaling and the FBF (for fem-3 binding factor) RNA-binding protein. Here, we report that the LIP-1 phosphatase, a proposed homolog of mitogen-activated protein (MAP) kinase phosphatases, is required for the normal extent of germline proliferation, and that lip-1 controls germline proliferation by regulating MAP kinase activity. In wild-type germ lines, LIP-1 protein is present in the proximal third of the mitotic region, consistent with its effect on germline proliferation. We provide evidence that lip-1 expression in the germline mitotic region is controlled by a combination of GLP-1/Notch signaling and FBF repression. Unexpectedly, FBF controls the accumulation of lip-1 mRNA, and therefore is likely to control its stability or 3'-end formation. In a sensitized mutant background, LIP-1 can function as a pivotal regulator of the decision between proliferation and differentiation. The control of germline proliferation by LIP-1 has intriguing parallels with the control of stem cells and progenitor cells in vertebrates.

3' Untranslated Regions↗
PubMed2005

Dose-dependent control of proliferation and sperm specification by FOG-1/CPEB.

RNA-binding proteins control germline development in metazoans. This work focuses on control of the C. elegans germline by two RNA-binding proteins: FOG-1, a CPEB homolog; and FBF, a PUF family member. Previous studies have shown that FOG-1 specifies the sperm fate and that FBF promotes proliferation. Here, we report that FOG-1 also promotes proliferation. Whereas fbf-1 fbf-2 double mutants make approximately 120 germ cells, fog-1; fbf-1 fbf-2 triple mutants make only approximately 10 germ cells. The triple mutant germline divides normally until early L2, when germ cells prematurely enter meiosis and begin oogenesis. Importantly, fog-1/+; fbf-1 fbf-2 animals make more germ cells than fbf-1 fbf-2 double mutants, demonstrating that one dose of wild-type fog-1 promotes proliferation more effectively than two doses - at least in the absence of FBF. FOG-1 protein is barely detectable in proliferating germ cells, but abundant in germ cells destined for spermatogenesis. Based on fog-1 dose effects, together with the gradient of FOG-1 protein abundance, we suggest that low FOG-1 promotes proliferation and high FOG-1 specifies spermatogenesis. FBF binds specifically to regulatory elements in the fog-1 3'UTR, and FOG-1 increases in animals lacking FBF. Therefore, FBF represses fog-1 expression. We suggest that FBF promotes continued proliferation, at least in part, by maintaining FOG-1 at a low level appropriate for proliferation. The dose-dependent control of proliferation and cell fate by FOG-1 has striking parallels with Xenopus CPEB, suggesting a conserved mechanism in animal development.

Animals↗
PubMed2005

A single spacer nucleotide determines the specificities of two mRNA regulatory proteins.

Regulation of messenger RNA is crucial in many contexts, including development, memory and cell growth. The 3' untranslated region is a rich repository of regulatory elements that bind proteins and microRNAs. Here we focus on PUF proteins, an important family of mRNA regulatory proteins crucial in stem-cell proliferation, pattern formation and synaptic plasticity. We show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. A discrete protein segment is responsible for the difference. We propose that a structural distortion in the central region of FBF imposes the requirement for the additional nucleotide and that this mode of PUF specificity may be common. We suggest that new specificities can be designed and selected using the PUF scaffold.

Animals↗
PubMed2005

Vertebrate GLD2 poly(A) polymerases in the germline and the brain.

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.

3T3 Cells↗
PubMed2005

Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1.

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.

Amino Acid Sequence↗
PubMed2004

RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening.

The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions. An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD). A productive RNA-protein interaction activates a reporter gene in vivo. The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence. However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined systematically. This limits interpretation of the data and complicates the development of new strategies. Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets. We compare activation of two reporter genes as a function of the in vitro affinity of the interaction. HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd. Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein. We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries. This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein. We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Mammalian GLD-2 homologs are poly(A) polymerases.

GLD-2 is a cytoplasmic poly(A) polymerase present in the Caenorhabditis elegans germ line and embryo. It is a divergent member of the DNA polymerase beta nucleotidyl transferase superfamily, which includes CCA-adding enzymes, DNA polymerases and eukaryotic nuclear poly(A) polymerases. The polyadenylation activity of GLD-2 is stimulated by physical interaction with an RNA binding protein, GLD-3. To test whether GLD-3 might stimulate GLD-2 by recruiting it to RNA, we tethered C. elegans GLD-2 to mRNAs in Xenopus oocytes by using MS2 coat protein. Tethered GLD-2 adds poly(A) and stimulates translation of the mRNA, demonstrating that recruitment is sufficient to stimulate polyadenylation activity. We use the same tethered assay to identify human and mouse poly(A) polymerases related to GLD-2. This may provide entrees to previously uncharacterized modes of polyadenylation in mammalian cells.

Animals↗
PubMed2004

FBF-1 and FBF-2 regulate the size of the mitotic region in the C. elegans germline.

In the C. elegans germline, GLP-1/Notch signaling and two nearly identical RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, we show that the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but that they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3'UTR repression. We propose that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development.

3' Untranslated Regions↗
PubMed2004

Tissue-specific modification of gld-2 mRNA in C. elegans: likely C-to-U editing.

Seventeen years after the discovery of tissue-specific apoB mRNA editing, only three nucleus-encoded mRNAs have been shown to undergo C-to-U editing. All three mRNAs occur in mammals. apoB mRNA editing is tissue-specific and occurs normally, whereas NF1 and NAT1 mRNA editing is found largely in tumors. Here we report the first example of C-to-U RNA editing in Caenorhabditis elegans. The gld-2 gene encodes an atypical poly(A) polymerase that governs the mitosis/meiosis decision in the germ line as well as progression through meiosis and early embryogenesis. At least two of its alternatively spliced transcripts are germline-specific. We find that most and perhaps all germline-specific transcripts generated by the gld-2 gene undergo C-to-U editing, but that somatic transcripts show no detectable editing. The gld-2 C-to-U editing event changes the codon from CCG to CUG, which is predicted to cause a proline to leucine substitution in the protein sequence. Our findings suggest the presence of a sequence- and tissue-specific cytidine deaminase acting on RNA, or CDAR. This CDAR modifies a specific base in gld-2 mRNA, and acts only in the germline.

Animals↗
PubMed2003

Regulation of the mitosis/meiosis decision in the Caenorhabditis elegans germline.

During the development of multicellular organisms, the processes of growth and differentiation are kept in balance to generate and maintain tissues and organs of the correct size, shape and cellular composition. We have investigated the molecular controls of growth and differentiation in the Caenorhabditis elegans germline. A single somatic cell, called the distal tip cell, promotes mitotic proliferation in the adjacent germline by GLP-1/Notch signalling. Within the germline, the decisions between mitosis and meiosis and between spermatogenesis and oogenesis are controlled by a group of conserved RNA regulators. FBF, a member of the PUF (for Pumilio and FBF) family of RNA-binding proteins, promotes mitosis by repressing gld-1 mRNA activity; the GLD-1, GLD-2, GLD-3 and NOS-3 proteins promote entry into meiosis by regulating mRNAs that remain unknown. The regulatory balance between opposing FBF and GLD activities is crucial for controlling the extent of germline proliferation. PUF proteins regulate germline stem cells in both Drosophila and C. elegans and are localized to germline stem cells of the mammalian testis. Therefore, this post-transcriptional regulatory switch may be an ancient mechanism for controlling maintenance of stem cells versus differentiation.

Animals↗